RETRACTED ARTICLE: High doses of garlic extract significantly attenuated the ratio of serum LDL to HDL level in rat-fed with hypercholesterolemia diet

Animals and animal ethics/and or welfare

Forty healthy male Wistar albino rats weighing 200–250 grams were obtained from the Animal Care Center at College of Medicine, Shiraz University. Animals were housed in wire cages(four per cage,and rats were kept in individual propylene cages under standard laboratory conditions by the dimensions of 30 × 50 × 25 cm3 two by two,1 month before the start of the experiments, and maintained under a daily controlled lighting cycle(12 h-light and 12 h-dark) at 22 + 2 °C and 60 % humidity with free access to rat diet and tap water for one week to adapt to the laboratory environment prior to the experiments. Animals were kept for 4 weeks to allow acclimatization to the animal facility before starting the experiments.

All animals received human care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health

Preparation of garlic extracts

Garlic bulbs utilized in these studies was purchased from a local grocery herb store in Shiraz, Iran. The garlic powder was prepared from fresh garlic bulb, which was heat-dried at 60–70 °C, and then ground by a mill. Volatile compounds in the garlic powder were analyzed by gas chromatography using daily disulfide as a standard [28, 29]. All garlic derivatives were stored at 4 °C and stocks of the water-soluble compounds were made fresh every time before use. Briefly, garlic cloves were peeled and homogenized with a small amount of quartz sand in 20 mmol/L Tris–HCl buffer (pH 7.4), and the debris was removed by centrifugation at 21,000 × g for 20 min at 25 °C. Subsequently, 30 g and 60 g of the powdered seeds were added to 400 ml and 800 ml of distilled water, and the extraction was obtained by steam distillation. The distillation process was continued until 200 mg/kg and 400 mg/kg of distillate were collected. The distillate was extracted three times with hydro-alcoholic liquid

Experimental protocol/or procedure and treatments

The rats were divided into four groups and randomly allotted into one of four experimental groups each group contained ten animals. The control group was fed a standard diet (normal control)(for first stage 15 days and second stage 30 days) (n = 10), and test animals in group II was fed a standard diet plus 10 % cholesterol-enriched high-fat (U.A.R., Paris, France; hypercholesterolemic controls) (for 2 weeks and 4 weeks) (n = 10);

Groups III and IV, the rats were fed with a lipogenic diet containing standard pellet diet supplemented with 0.5 % (w/w) cholic acid, 20 % (w/w) sunflower oil and 2 % (w/w) cholesterol for at least two weeks to produce hyperlipidemia. Additionally, groups III and IV drank water containing 3 % (v/v) ethanol). Groups III and IV received garlic extract as an intraperitoneal (IP) dose of 200 and 400 mg/kg/bw (for 2 and 4 weeks) (garlic diet, n = 10), respectively. In addition, the garlic extract in experimental group was injected intraperitoneally. Blood samples were collected into test tubes containing EDTA through cardiac puncture. The plasma samples were separated by low speed centrifugation (2000 g) for 10 min and were stored at _80 _C until they were analyzed. All animal procedures were performed with regard to Iranian animal ethics society and local university rules.

Plasma biochemical measurements/and or biochemical assay of serum lipids

Blood samples were taken directly from the heart in a centrifuge tube and allowed to form serum and processed as previously described, however, in each rat was determined after the start the treatment and every 15 d in total 2 times, and or can be expressed that after the 4-weeks treatment, the rats were killed using urethane anesthesia. The blood was collected by cardiac puncture and allowed to clot, and the clotted blood was then centrifuged at 4500 × g for 10 min. The serum was separated and stored at −80 °C for HDL and LDL analysis, and these serum concentrations were determined with commercially available enzyme kits (BioMerieux, Marcy, France).

Statistical analysis

All data are presented as means ± SEM. Statistical analyses were analyzed using one-way ANOVA and two-way ANOVA, and significant differences between means were evaluated by the Tukey’s range post-hoc test compare between experimental groups. Differences with P < 0.05 were considered significant.